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1.
Nucleic Acids Res ; 49(2): 954-968, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33398345

RESUMO

Catalytically inactive Cas9 (dCas9) has become an increasingly popular tool for targeted gene activation/inactivation, live-cell imaging, and base editing. While dCas9 was reported to induce base substitutions and indels, it has not been associated with structural variations. Here, we show that dCas9 impedes replication fork progression to destabilize tandem repeats in budding yeast. When targeted to the CUP1 array comprising ∼16 repeat units, dCas9 induced its contraction in most cells, especially in the presence of nicotinamide. Replication intermediate analysis demonstrated replication fork stalling in the vicinity of dCas9-bound sites. Genetic analysis indicated that while destabilization is counteracted by the replisome progression complex components Ctf4 and Mrc1 and the accessory helicase Rrm3, it involves single-strand annealing by the recombination proteins Rad52 and Rad59. Although dCas9-mediated replication fork stalling is a potential risk in conventional applications, it may serve as a novel tool for both mechanistic studies and manipulation of genomic instability.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Replicação do DNA , DNA Fúngico/genética , Instabilidade Genômica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteína 9 Associada à CRISPR/química , Variações do Número de Cópias de DNA , DNA Fúngico/metabolismo , DNA de Cadeia Simples/genética , Edição de Genes , Genes Fúngicos , Metalotioneína/genética , Mutagênese , Niacinamida/farmacologia , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Sequências de Repetição em Tandem
2.
PLoS Genet ; 16(12): e1009256, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33370257

RESUMO

Endogenous genotoxic stress occurs in healthy cells due to competition between DNA replication machinery, and transcription and topographic relaxation processes. This causes replication fork stalling and regression, which can further collapse to form single-ended double strand breaks (seDSBs). Super-resolution microscopy has made it possible to directly observe replication stress and DNA damage inside cells, however new approaches to sample preparation and analysis are required. Here we develop and apply multicolor single molecule microscopy to visualize individual replication forks under mild stress from the trapping of Topoisomerase I cleavage complexes, a damage induction which closely mimics endogenous replicative stress. We observe RAD51 and RAD52, alongside RECQ1, as the first responder proteins to stalled but unbroken forks, whereas Ku and MRE11 are initially recruited to seDSBs. By implementing novel super-resolution imaging assays, we are thus able to discern closely related replication fork stress motifs and their repair pathways.


Assuntos
Quebras de DNA de Cadeia Dupla , Replicação do DNA , DNA/química , Imagem Individual de Molécula/métodos , Linhagem Celular Tumoral , DNA/genética , Humanos , Proteína Homóloga a MRE11/metabolismo , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , RecQ Helicases/metabolismo
3.
PLoS Genet ; 16(12): e1008603, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33370275

RESUMO

Telomeres have the ability to adopt a lariat conformation and hence, engage in long and short distance intra-chromosome interactions. Budding yeast telomeres were proposed to fold back into subtelomeric regions, but a robust assay to quantitatively characterize this structure has been lacking. Therefore, it is not well understood how the interactions between telomeres and non-telomeric regions are established and regulated. We employ a telomere chromosome conformation capture (Telo-3C) approach to directly analyze telomere folding and its maintenance in S. cerevisiae. We identify the histone modifiers Sir2, Sin3 and Set2 as critical regulators for telomere folding, which suggests that a distinct telomeric chromatin environment is a major requirement for the folding of yeast telomeres. We demonstrate that telomeres are not folded when cells enter replicative senescence, which occurs independently of short telomere length. Indeed, Sir2, Sin3 and Set2 protein levels are decreased during senescence and their absence may thereby prevent telomere folding. Additionally, we show that the homologous recombination machinery, including the Rad51 and Rad52 proteins, as well as the checkpoint component Rad53 are essential for establishing the telomere fold-back structure. This study outlines a method to interrogate telomere-subtelomere interactions at a single unmodified yeast telomere. Using this method, we provide insights into how the spatial arrangement of the chromosome end structure is established and demonstrate that telomere folding is compromised throughout replicative senescence.


Assuntos
Replicação do DNA , Histona Desacetilases/metabolismo , Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2/metabolismo , Telômero/genética , Histona Desacetilases/genética , Metiltransferases/genética , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas Repressoras/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Sirtuína 2/genética , Telômero/química , Homeostase do Telômero
4.
Proc Natl Acad Sci U S A ; 117(28): 16527-16536, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601218

RESUMO

Folate deprivation drives the instability of a group of rare fragile sites (RFSs) characterized by CGG trinucleotide repeat (TNR) sequences. Pathological expansion of the TNR within the FRAXA locus perturbs DNA replication and is the major causative factor for fragile X syndrome, a sex-linked disorder associated with cognitive impairment. Although folate-sensitive RFSs share many features with common fragile sites (CFSs; which are found in all individuals), they are induced by different stresses and share no sequence similarity. It is known that a pathway (termed MiDAS) is employed to complete the replication of CFSs in early mitosis. This process requires RAD52 and is implicated in generating translocations and copy number changes at CFSs in cancers. However, it is unclear whether RFSs also utilize MiDAS and to what extent the fragility of CFSs and RFSs arises by shared or distinct mechanisms. Here, we demonstrate that MiDAS does occur at FRAXA following folate deprivation but proceeds via a pathway that shows some mechanistic differences from that at CFSs, being dependent on RAD51, SLX1, and POLD3. A failure to complete MiDAS at FRAXA leads to severe locus instability and missegregation in mitosis. We propose that break-induced DNA replication is required for the replication of FRAXA under folate stress and define a cellular function for human SLX1. These findings provide insights into how folate deprivation drives instability in the human genome.


Assuntos
Endodesoxirribonucleases/metabolismo , Ácido Fólico/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Mitose , Rad51 Recombinase/metabolismo , DNA/genética , DNA/metabolismo , Reparo do DNA , Endodesoxirribonucleases/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/fisiopatologia , Humanos , Rad51 Recombinase/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinases/genética , Recombinases/metabolismo
5.
Nat Commun ; 11(1): 3181, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576832

RESUMO

The DNA damage checkpoint (DDC) is often robustly activated during the homologous recombination (HR) repair of DNA double strand breaks (DSBs). DDC activation controls several HR repair factors by phosphorylation, preventing premature segregation of entangled chromosomes formed during HR repair. The DDC mediator 53BP1/Rad9 limits the nucleolytic processing (resection) of a DSB, controlling the formation of the 3' single-stranded DNA (ssDNA) filament needed for recombination, from yeast to human. Here we show that Rad9 promotes stable annealing between the recombinogenic filament and the donor template in yeast, limiting strand rejection by the Sgs1 and Mph1 helicases. This regulation allows repair by long tract gene conversion, crossover recombination and break-induced replication (BIR), only after DDC activation. These findings shed light on how cells couple DDC with the choice and effectiveness of HR sub-pathways, with implications for genome instability and cancer.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Reparo do DNA/genética , Reparo do DNA/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Proteínas de Ciclo Celular/genética , Sobrevivência Celular , RNA Helicases DEAD-box/metabolismo , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples , Proteínas de Ligação a DNA/metabolismo , Conversão Gênica , Instabilidade Genômica , Recombinação Homóloga , Humanos , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , RecQ Helicases/metabolismo , Reparo de DNA por Recombinação , Proteínas de Saccharomyces cerevisiae/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
6.
Nat Commun ; 11(1): 2834, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503981

RESUMO

Recruitment of DNA repair proteins to DNA damage sites is a critical step for DNA repair. Post-translational modifications of proteins at DNA damage sites serve as DNA damage codes to recruit specific DNA repair factors. Here, we show that mRNA is locally modified by m5C at sites of DNA damage. The RNA methyltransferase TRDMT1 is recruited to DNA damage sites to promote m5C induction. Loss of TRDMT1 compromises homologous recombination (HR) and increases cellular sensitivity to DNA double-strand breaks (DSBs). In the absence of TRDMT1, RAD51 and RAD52 fail to localize to sites of reactive oxygen species (ROS)-induced DNA damage. In vitro, RAD52 displays an increased affinity for DNA:RNA hybrids containing m5C-modified RNA. Loss of TRDMT1 in cancer cells confers sensitivity to PARP inhibitors in vitro and in vivo. These results reveal an unexpected TRDMT1-m5C axis that promotes HR, suggesting that post-transcriptional modifications of RNA can also serve as DNA damage codes to regulate DNA repair.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Quebras de DNA de Cadeia Dupla , Recombinação Homóloga , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/metabolismo , Animais , Linhagem Celular Tumoral , Citosina/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Resistencia a Medicamentos Antineoplásicos/genética , Técnicas de Silenciamento de Genes , Humanos , Metilação , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , RNA Interferente Pequeno/metabolismo , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Nat Struct Mol Biol ; 27(5): 424-437, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32398827

RESUMO

Oncogene activation during tumorigenesis generates DNA replication stress, a known driver of genome rearrangements. In response to replication stress, certain loci, such as common fragile sites and telomeres, remain under-replicated during interphase and subsequently complete locus duplication in mitosis in a process known as 'MiDAS'. Here, we demonstrate that RTEL1 (regulator of telomere elongation helicase 1) has a genome-wide role in MiDAS at loci prone to form G-quadruplex-associated R-loops, in a process that is dependent on its helicase function. We reveal that SLX4 is required for the timely recruitment of RTEL1 to the affected loci, which in turn facilitates recruitment of other proteins required for MiDAS, including RAD52 and POLD3. Our findings demonstrate that RTEL1 is required for MiDAS and suggest that RTEL1 maintains genome stability by resolving conflicts that can arise between the replication and transcription machineries.


Assuntos
DNA Helicases/genética , DNA Helicases/metabolismo , Quadruplex G , Genoma Humano/genética , Mitose , Animais , Linhagem Celular , DNA Helicases/química , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Instabilidade Genômica , Humanos , Imunoprecipitação , Camundongos , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Conformação de Ácido Nucleico , RNA Helicases/genética , RNA Helicases/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinases/genética , Recombinases/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo
8.
PLoS Genet ; 16(2): e1008608, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32012161

RESUMO

DNA double-strand breaks (DSBs) are toxic forms of DNA damage that must be repaired to maintain genome integrity. Telomerase can act upon a DSB to create a de novo telomere, a process that interferes with normal repair and creates terminal deletions. We previously identified sequences in Saccharomyces cerevisiae (SiRTAs; Sites of Repair-associated Telomere Addition) that undergo unusually high frequencies of de novo telomere addition, even when the original chromosome break is several kilobases distal to the eventual site of telomerase action. Association of the single-stranded telomere binding protein Cdc13 with a SiRTA is required to stimulate de novo telomere addition. Because extensive resection must occur prior to Cdc13 binding, we utilized these sites to monitor the effect of proteins involved in homologous recombination. We find that telomere addition is significantly reduced in the absence of the Rad51 recombinase, while loss of Rad52, required for Rad51 nucleoprotein filament formation, has no effect. Deletion of RAD52 suppresses the defect of the rad51Δ strain, suggesting that Rad52 inhibits de novo telomere addition in the absence of Rad51. The ability of Rad51 to counteract this effect of Rad52 does not require DNA binding by Rad51, but does require interaction between the two proteins, while the inhibitory effect of Rad52 depends on its interaction with Replication Protein A (RPA). Intriguingly, the genetic interactions we report between RAD51 and RAD52 are similar to those previously observed in the context of checkpoint adaptation. Forced recruitment of Cdc13 fully restores telomere addition in the absence of Rad51, suggesting that Rad52, through its interaction with RPA-coated single-stranded DNA, inhibits the ability of Cdc13 to bind and stimulate telomere addition. Loss of the Rad51-Rad52 interaction also stimulates a subset of Rad52-dependent microhomology-mediated repair (MHMR) events, consistent with the known ability of Rad51 to prevent single-strand annealing.


Assuntos
Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Reparo de DNA por Recombinação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Telômero/metabolismo , Quebras de DNA de Cadeia Dupla , Técnicas de Inativação de Genes , Mutação , Ligação Proteica/genética , Rad51 Recombinase/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/metabolismo
9.
Nat Commun ; 11(1): 695, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-32019927

RESUMO

Cellular processes are influenced by liquid phase separation, but its role in DNA repair is unclear. Here, we show that in Saccharomyces cerevisiae, liquid droplets made up of DNA repair proteins cooperate with different types of DNA damage-inducible intranuclear microtubule filaments (DIMs) to promote the clustering of DNA damage sites and maintain genome stability. Rad52 DNA repair proteins at different DNA damage sites assemble in liquid droplets that fuse into a repair centre droplet via the action of petite DIMs (pti-DIMs). This larger droplet concentrates tubulin and projects short aster-like DIMs (aster-DIMs), which tether the repair centre to longer DIMs mediating the mobilization of damaged DNA to the nuclear periphery for repair. Our findings indicate that cooperation between Rad52 liquid droplets and various types of nuclear filaments promotes the assembly and function of the DNA repair centre.


Assuntos
Reparo do DNA , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Dano ao DNA , DNA Fúngico/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
10.
Sci Adv ; 6(6): eaay2669, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32083180

RESUMO

Homologous recombination is exquisitely activated only during specific cell phases. In the G1 phase, homologous recombination activity is completely suppressed. According to previous reports, the activation of homologous recombination during specific cell phases depends on the kinase activity of cyclin-dependent kinase 1 (CDK1). However, the precise regulatory mechanism and target substrates of CDK1 for this regulation have not been completely determined. Here, we report that the budding yeast CDK1, Cdc28, phosphorylates the major homologous recombination regulators Rad51 and Rad52. This phosphorylation occurs in the G2/M phase by Cdc28 in combination with G2/M phase cyclins. Nonphosphorylatable mutations in Rad51 and Rad52 impair the DNA binding affinity of Rad51 and the affinity between Rad52 rings that leads to their interaction. Collectively, our data provide detailed insights into the regulatory mechanism of cell cycle-dependent homologous recombination activation in eukaryotic cells.


Assuntos
Proteína Quinase CDC2/metabolismo , Ciclo Celular/genética , Recombinação Homóloga , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Ativação Transcricional , CDC2-CDC28 Quinases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fosforilação
11.
Mol Cell ; 77(3): 528-541.e8, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-31759821

RESUMO

Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcription-replication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase δ, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends on MUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution.


Assuntos
Replicação do DNA/fisiologia , Estruturas R-Loop/genética , Rad51 Recombinase/metabolismo , Linhagem Celular Tumoral , DNA Ligases/metabolismo , DNA Polimerase III/metabolismo , Replicação do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Células HeLa , Humanos , Estruturas R-Loop/fisiologia , Rad51 Recombinase/genética , Rad51 Recombinase/fisiologia , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , RecQ Helicases/metabolismo , RecQ Helicases/fisiologia , Transcrição Genética/genética
12.
Aging Cell ; 19(2): e13068, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31833215

RESUMO

Maf1 is the master repressor of RNA polymerase III responsible for transcription of tRNAs and 5S rRNAs. Maf1 is negatively regulated via phosphorylation by the mTOR pathway, which governs protein synthesis, growth control, and lifespan regulation in response to nutrient availability. Inhibiting the mTOR pathway extends lifespan in various organisms. However, the downstream effectors for the regulation of cell homeostasis that are critical to lifespan extension remain elusive. Here we show that fission yeast Maf1 is required for lifespan extension. Maf1's function in tRNA repression is inhibited by mTOR-dependent phosphorylation, whereas Maf1 is activated via dephosphorylation by protein phosphatase complexes, PP4 and PP2A. Mutational analysis reveals that Maf1 phosphorylation status influences lifespan, which is correlated with elevated tRNA and protein synthesis levels in maf1∆ cells. However, mTOR downregulation, which negates protein synthesis, fails to rescue the short lifespan of maf1∆ cells, suggesting that elevated protein synthesis is not a cause of lifespan shortening in maf1∆ cells. Interestingly, maf1∆ cells accumulate DNA damage represented by formation of Rad52 DNA damage foci and Rad52 recruitment at tRNA genes. Loss of the Rad52 DNA repair protein further exacerbates the shortened lifespan of maf1∆ cells. Strikingly, PP4 deletion alleviates DNA damage and rescues the short lifespan of maf1∆ cells even though tRNA synthesis is increased in this condition, suggesting that elevated DNA damage is the major cause of lifespan shortening in maf1∆ cells. We propose that Maf1-dependent inhibition of tRNA synthesis controls fission yeast lifespan by preventing genomic instability that arises at tRNA genes.


Assuntos
Regulação Fúngica da Expressão Gênica , Instabilidade Genômica/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , RNA de Transferência/genética , Proteínas Repressoras/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Imunoprecipitação da Cromatina , Dano ao DNA/genética , Glucose/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , RNA de Transferência/biossíntese , RNA de Transferência/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas Repressoras/genética , Schizosaccharomyces/metabolismo , Schizosaccharomyces/fisiologia , Proteínas de Schizosaccharomyces pombe/genética
13.
PLoS Genet ; 15(10): e1008441, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31584934

RESUMO

Replication stress poses a serious threat to genome stability. Recombination-Dependent-Replication (RDR) promotes DNA synthesis resumption from arrested forks. Despite the identification of chromatin restoration pathways after DNA repair, crosstalk coupling RDR and chromatin assembly is largely unexplored. The fission yeast Chromatin Assembly Factor-1, CAF-1, is known to promote RDR. Here, we addressed the contribution of histone deposition to RDR. We expressed a mutated histone, H3-H113D, to genetically alter replication-dependent chromatin assembly by destabilizing (H3-H4)2 tetramer. We established that DNA synthesis-dependent histone deposition, by CAF-1 and Asf1, promotes RDR by preventing Rqh1-mediated disassembly of joint-molecules. The recombination factor Rad52 promotes CAF-1 binding to sites of recombination-dependent DNA synthesis, indicating that histone deposition occurs downstream Rad52. Histone deposition and Rqh1 activity act synergistically to promote cell resistance to camptothecin, a topoisomerase I inhibitor that induces replication stress. Moreover, histone deposition favors non conservative recombination events occurring spontaneously in the absence of Rqh1, indicating that the stabilization of joint-molecules by histone deposition also occurs independently of Rqh1 activity. These results indicate that histone deposition plays an active role in promoting RDR, a benefit counterbalanced by stabilizing at-risk joint-molecules for genome stability.


Assuntos
Montagem e Desmontagem da Cromatina , Replicação do DNA , Instabilidade Genômica , Histonas/metabolismo , Recombinação Genética , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/metabolismo , Histonas/genética , Chaperonas Moleculares/metabolismo , Mutação , Multimerização Proteica/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Ribonucleases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
14.
Genetics ; 213(3): 819-834, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31533921

RESUMO

The accuracy of most DNA processes depends on chromatin integrity and dynamics. Our analyses in the yeast Saccharomyces cerevisiae show that an absence of Swr1 (the catalytic and scaffold subunit of the chromatin-remodeling complex SWR) leads to the formation of long-duration Rad52, but not RPA, foci and to an increase in intramolecular recombination. These phenotypes are further increased by MMS, zeocin, and ionizing radiation, but not by double-strand breaks, HU, or transcription/replication collisions, suggesting that they are associated with specific DNA lesions. Importantly, these phenotypes can be specifically suppressed by mutations in: (1) chromatin-anchorage internal nuclear membrane components (mps3∆75-150 and src1∆); (2) actin and actin regulators (act1 -157, act1 -159, crn1∆, and cdc42 -6); or (3) the SWR subunit Swc5 and the SWR substrate Htz1 However, they are not suppressed by global disruption of actin filaments or by the absence of Csm4 (a component of the external nuclear membrane that forms a bridging complex with Mps3, thus connecting the actin cytoskeleton with chromatin). Moreover, swr1∆-induced Rad52 foci and intramolecular recombination are not associated with tethering recombinogenic DNA lesions to the nuclear periphery. In conclusion, the absence of Swr1 impairs efficient recombinational repair of specific DNA lesions by mechanisms that are influenced by SWR subunits, including actin, and nuclear envelope components. We suggest that these recombinational phenotypes might be associated with a pathological effect on homologous recombination of actin-containing complexes.


Assuntos
Citoesqueleto de Actina/metabolismo , Adenosina Trifosfatases/genética , Recombinação Homóloga , Membrana Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Citoesqueleto de Actina/genética , Actinas/genética , Actinas/metabolismo , Adenosina Trifosfatases/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Membrana Nuclear/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/genética , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/metabolismo
15.
Mol Cell ; 76(5): 699-711.e6, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31542296

RESUMO

Rad52 is a key factor for homologous recombination (HR) in yeast. Rad52 helps assemble Rad51-ssDNA nucleoprotein filaments that catalyze DNA strand exchange, and it mediates single-strand DNA annealing. We find that Rad52 has an even earlier function in HR in restricting DNA double-stranded break ends resection that generates 3' single-stranded DNA (ssDNA) tails. In fission yeast, Exo1 is the primary resection nuclease, with the helicase Rqh1 playing a minor role. We demonstrate that the choice of two extensive resection pathways is regulated by Rad52. In rad52 cells, the resection rate increases from ∼3-5 kb/h up to ∼10-20 kb/h in an Rqh1-dependent manner, while Exo1 becomes dispensable. Budding yeast Rad52 similarly inhibits Sgs1-dependent resection. Single-molecule analysis with purified budding yeast proteins shows that Rad52 competes with Sgs1 for DNA end binding and inhibits Sgs1 translocation along DNA. These results identify a role for Rad52 in limiting ssDNA generated by end resection.


Assuntos
Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Reparo do DNA , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Regulação Fúngica da Expressão Gênica , Cinética , Mutação , Domínios Proteicos , Transporte Proteico , Proteína Rad52 de Recombinação e Reparo de DNA/genética , RecQ Helicases/genética , RecQ Helicases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética
16.
Mol Biol Cell ; 30(21): 2620-2625, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31483739

RESUMO

During S phase in Saccharomyces cerevisiae, chromosomal loci become mobile in response to DNA double-strand breaks both at the break site (local mobility) and throughout the nucleus (global mobility). Increased nuclear exploration is regulated by the recombination machinery and the DNA damage checkpoint and is likely an important aspect of homology search. While mobility in response to DNA damage has been studied extensively in S phase, the response in interphase has not, and the question of whether homologous recombination proceeds to completion in G1 phase remains controversial. Here, we find that global mobility is triggered in G1 phase. As in S phase, global mobility in G1 phase is controlled by the DNA damage checkpoint and the Rad51 recombinase. Interestingly, despite the restriction of Rad52 mediator foci to S phase, Rad51 foci form at high levels in G1 phase. Together, these observations indicate that the recombination and checkpoint machineries promote global mobility in G1 phase, supporting the notion that recombination can occur in interphase diploids.


Assuntos
Núcleo Celular/genética , Cromossomos Fúngicos/genética , Dano ao DNA , Fase G1/genética , Saccharomyces cerevisiae/genética , Núcleo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Recombinação Homóloga , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Fase S/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
17.
PLoS Genet ; 15(8): e1008319, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31381562

RESUMO

Disrupting either the DNA annealing factor RAD52 or the A-family DNA polymerase POLQ can cause synthetic lethality with defects in BRCA1 and BRCA2, which are tumor suppressors important for homology-directed repair of DNA double-strand breaks (DSBs), and protection of stalled replication forks. A likely mechanism of this synthetic lethality is that RAD52 and/or POLQ are important for backup pathways for DSB repair and/or replication stress responses. The features of DSB repair events that require RAD52 vs. POLQ, and whether combined disruption of these factors causes distinct effects on genome maintenance, have been unclear. Using human U2OS cells, we generated a cell line with POLQ mutations upstream of the polymerase domain, a RAD52 knockout cell line, and a line with combined disruption of both genes. We also examined RAD52 and POLQ using RNA-interference. We find that combined disruption of RAD52 and POLQ causes at least additive hypersensitivity to cisplatin, and a synthetic reduction in replication fork restart velocity. We also examined the influence of RAD52 and POLQ on several DSB repair events. We find that RAD52 is particularly important for repair using ≥ 50 nt repeat sequences that flank the DSB, and that also involve removal of non-homologous sequences flanking the repeats. In contrast, POLQ is important for repair events using 6 nt (but not ≥ 18 nt) of flanking repeats that are at the edge of the break, as well as oligonucleotide microhomology-templated (i.e., 12-20 nt) repair events requiring nascent DNA synthesis. Finally, these factors show key distinctions with BRCA2, regarding effects on DSB repair events and response to stalled replication forks. These findings indicate that RAD52 and POLQ have distinct roles in genome maintenance, including for specific features of DSB repair events, such that combined disruption of these factors may be effective for genotoxin sensitization and/or synthetic lethal strategies.


Assuntos
DNA Polimerase Dirigida por DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Reparo de DNA por Recombinação , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Mutação , Neoplasias/tratamento farmacológico , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Mutações Sintéticas Letais
18.
Mol Cell ; 76(1): 11-26.e7, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31400850

RESUMO

Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in aggressive cancers. We show that the disruption of RAD51-associated protein 1 (RAD51AP1) in ALT+ cancer cells leads to generational telomere shortening. This is due to RAD51AP1's involvement in RAD51-dependent homologous recombination (HR) and RAD52-POLD3-dependent break induced DNA synthesis. RAD51AP1 KO ALT+ cells exhibit telomere dysfunction and cytosolic telomeric DNA fragments that are sensed by cGAS. Intriguingly, they activate ULK1-ATG7-dependent autophagy as a survival mechanism to mitigate DNA damage and apoptosis. Importantly, RAD51AP1 protein levels are elevated in ALT+ cells due to MMS21 associated SUMOylation. Mutation of a single SUMO-targeted lysine residue perturbs telomere dynamics. These findings indicate that RAD51AP1 is an essential mediator of the ALT mechanism and is co-opted by post-translational mechanisms to maintain telomere length and ensure proliferation of ALT+ cancer cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Neoplasias/metabolismo , Proteínas de Ligação a RNA/metabolismo , Homeostase do Telômero , Telômero/metabolismo , Autofagia , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proliferação de Células , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Recombinação Homóloga , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligases/genética , Ligases/metabolismo , Lisina , Neoplasias/genética , Neoplasias/patologia , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Estabilidade Proteica , Proteínas de Ligação a RNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Transdução de Sinais , Sumoilação , Telômero/genética , Telômero/patologia
19.
Genes Dev ; 33(13-14): 814-827, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171703

RESUMO

Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. One of the hallmarks of ALT cancer is the excessively clustered telomeres in promyelocytic leukemia (PML) bodies, represented as large bright telomere foci. Here, we present a model system that generates telomere clustering in nuclear polySUMO (small ubiquitin-like modification)/polySIM (SUMO-interacting motif) condensates, analogous to PML bodies, and thus artificially engineered ALT-associated PML body (APB)-like condensates in vivo. We observed that the ALT-like phenotypes (i.e., a small fraction of heterogeneous telomere lengths and formation of C circles) are rapidly induced by introducing the APB-like condensates together with BLM through its helicase domain, accompanied by ssDNA generation and RPA accumulation at telomeres. Moreover, these events lead to mitotic DNA synthesis (MiDAS) at telomeres mediated by RAD52 through its highly conserved N-terminal domain. We propose that the clustering of large amounts of telomeres in human cancers promotes ALT that is mediated by MiDAS, analogous to Saccharomyces cerevisiae type II ALT survivors.


Assuntos
Núcleo Celular/metabolismo , DNA/biossíntese , Leucemia Promielocítica Aguda/fisiopatologia , Mitose , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , RecQ Helicases/metabolismo , Homeostase do Telômero/genética , Motivos de Aminoácidos , Linhagem Celular Tumoral , Expressão Gênica , Humanos , Leucemia Promielocítica Aguda/genética , Fenótipo , Transporte Proteico , Proteína SUMO-1/metabolismo , Telômero/genética , Telômero/metabolismo
20.
Mol Cancer Res ; 17(8): 1687-1698, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31113828

RESUMO

Mitotic DNA synthesis is a recently discovered mechanism that resolves late replication intermediates, thereby supporting cell proliferation under replication stress. This unusual form of DNA synthesis occurs in the absence of RAD51 or BRCA2, which led to the identification of RAD52 as a key player in this process. Notably, mitotic DNA synthesis is predominantly observed at chromosome loci that colocalize with FANCD2 foci. However, the role of this protein in mitotic DNA synthesis remains largely unknown. In this study, we investigated the role of FANCD2 and its interplay with RAD52 in mitotic DNA synthesis using aphidicolin as a universal inducer of this process. After examining eight human cell lines, we provide evidence for FANCD2 rather than RAD52 as a fundamental supporter of mitotic DNA synthesis. In cancer cell lines, FANCD2 exerts this role independently of RAD52. Surprisingly, RAD52 is dispensable for mitotic DNA synthesis in noncancerous cell lines, but these cells strongly depend on FANCD2 for this process. Therefore, RAD52 functions selectively in cancer cells as a secondary regulator in addition to FANCD2 to facilitate mitotic DNA synthesis. As an alternative to aphidicolin, we found partial inhibition of origin licensing as an effective way to induce mitotic DNA synthesis preferentially in cancer cells. Importantly, cancer cells still perform mitotic DNA synthesis by dual regulation of FANCD2 and RAD52 under such conditions. IMPLICATIONS: These key differences in mitotic DNA synthesis between cancer and noncancerous cells advance our understanding of this mechanism and can be exploited for cancer therapies.


Assuntos
DNA/biossíntese , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Fibroblastos/citologia , Mitose , Neoplasias/patologia , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Epitélio Pigmentado da Retina/citologia , Núcleo Celular , Células Cultivadas , Dano ao DNA , Reparo do DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Fibroblastos/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Epitélio Pigmentado da Retina/metabolismo
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